Fungi within the Fusarium oxysporum species complex can cause root rot, seedling blight, and wilt of soybean. Isolates recovered from soybean vary in aggressiveness and also the type of symptoms they produce. The aim of this study was to identify genetic markers to detect aggressive soybean wilt isolates. Eighty isolates collected primarily from soybean were tested in the greenhouse for their ability to produce wilt symptoms using susceptible 'Jack' soybean. The same 80 isolates were assessed for the presence of fungal effector genes Fmk1, Fow1, Pda1, PelA, PelD, Pep1, Prt1, Rho1, Sge1, Six1, Six6, and Snf1. All polymerase chain reaction amplicons were sequenced, phylogenies were inferred, and analysis of molecular variance (AMOVA) was performed for 10 of the 12 genes. High incidence of vascular discoloration of roots or stems was observed with 3 isolates, while moderate to low levels of incidence were observed for 25 isolates. Fungal effector genes Fmk1, Fow1, PelA, Rho1, Sge1, and Snf1 were present in all isolates screened, while Pda1, PelD, Pep1, Prt1, Six1, and Six6 were dispersed among isolates. The Bayesian and AMOVA analyses found that the genes Fmk1, Fow1, Pda1, PelA, Rho1, Sge1, and Snf1 corresponded to previously designated clades based on tef1α and mitochondrial small subunit sequences. None of the genes had a significant association with wilt symptoms on soybean. Interestingly, the Six6 gene was only present in three previously known wilt isolates from soybean, common bean, and tomato; of these, the soybean and common bean isolates produced high levels of vascular wilt in our study.